Validated methods for phage selection, host range expansion, and lytic activity determination are indispensable for maximizing phage therapy outcomes. In this review, we describe some relevant methods, highlighting their advantages and disadvantages, and categorize them as preliminary or confirmatory methods where appropriate. Experimental conditions, such as the composition and consistency of culture media, have an impact on bacterial growth and, consequently, phage propagation and the selection of phage-resistant mutants. The phages require different experimental conditions to be tested to fully reveal their characteristics and phage therapy potential in view of their future use in therapy. Phage lytic activity or virulence should be considered as a result of the phage, its host, and intracellular/environmental factors, including the ability of a phage to recognize receptors on the bacterial cell surface. In vitro quantitative and qualitative measurements of phage characteristics, further validated by in vivo experiments, could be incorporated into one system or mathematical model/formula, which could predict a potential successful outcome of clinical applications.

As the global burden of disease caused by multidrug resistant bacteria is a major source of concern, credible clinical alternatives to antibiotic therapy, such as personalized phage therapy, are actively explored. Although phage therapy has been used for more than a century, the issue of an easy to implement diagnostic tool for determining phage susceptibility that meets current routine clinical needs is still open. In this Review, we summarize the existing methods used for determining phage activity on bacteria, including the three reference methods: the spot test, the double agar overlay plaque assay, and the Appelmans method. The first two methods rely on the principle of challenging the overnight growth of a lawn of bacteria in an agar matrix to a known relative phage to bacteria concentration and represent good screening tools to determine if the tested phage can be used for a “passive” and or “active” treatment. Beside these methods, several techniques, based on “real-time” growth kinetics assays (GKA) have been developed or are under development. They all monitor the growth of clinical isolates in the presence of phages, but use various detection methods, from classical optical density to more sophisticated techniques such as computer-assisted imagery, flow-cytometry, quantitative real-time polymerase chain reaction (qPCR) or metabolic indicators. Practical considerations as well as information provided about phage activity are reviewed for each technique. Finally, we also discuss the analytical and interpretative requirements for the implementation of a phage susceptibility testing tool in routine clinical microbiology.

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In the present study, we evaluated the effect of spray-drying formulations and operating parameters of a laboratory-scale spray-dryer on the characteristics of spray-dried powders containing two Pseudomonas aeruginosa bacteriophages exhibiting different morphotypes: a podovirus (LUZ19) and a myovirus (14-1). We optimized the production process for bacteriophage-loaded powders, with an emphasis on long-term storage under ICH (international conference on harmonization) conditions. D-trehalose-/L-isoleucine-containing bacteriophage mixtures were spray-dried from aqueous solutions using a Büchi Mini Spray-dryer B-290 (Flawil, Switzerland). A response surface methodology was used for the optimization of the spray-drying process, with the following as-evaluated parameters: Inlet temperature, spray gas flow rate, and the D-trehalose/L-isoleucine ratio. The dried powders were characterized in terms of yield, residual moisture content, and bacteriophage lytic activity. L-isoleucine has demonstrated a positive impact on the activity of LUZ19, but a negative impact on 14-1. We observed a negligible impact of the inlet temperature and a positive correlation of the spray gas flow rate with bacteriophage activity. After optimization, we were able to obtain dry powder preparations of both bacteriophages, which were stable for a minimum of one year under different ICH storage conditions (up to and including 40 °C and 75% relative humidity).